The specific aims of this project are threefold: The biological activity of human CRF (hrCRF) will be studied in rat and human tissues. The responses will be compared to those obtained with ovine CRF. Activity will be examined in vitro in a dispersed cell system derived from rat anterior pituitary and in vivo (a) in non-anesthetized freely moving adult male rats and monkeys with chronically implanted indwelling intra-atrial cannulae; (b) in normal human volunteers and in patients with disorders of the hypothalamic-pituitary-adrenal axis. In addition, the activity of selected putative synthetic CRF agonists will be examined in vitro. (2) CRF will be measured and characterized in the rat, sheep and human hypothalamus, in non-hypothalamic brain and extra-brain tissue, in lumbar CSF and peripheral blood in human and in hypophyseal portal blood and peripheral blood of the rat. The CRF content will be measured by radioimmunoassay, immunocytochemical and bioassay systems and chemical characterization of bioassayable CRF and CRF-LI will be accomplished by protein chemistry. (3) Finally, biosynthesis and secretion of CRF will be studied in the rat, sheep and man. The presence of immunologically identifiable precursor(s) to CRF in ovine, rat and human tissue will be studied by radioimmunoassay and by immunocytochemical staining utilizing antisera to portions of the sequence of the pre-pro CRF molecule in man and sheep. In vitro systems for examining biosynthesis will be standardized in ovine, rat and human hypothalamic tissue initially by in vitro pulse-chase procedures with tissue followed by immunoprecipitation as well as cell free translation. The species specific mRNA encoding the hrCRF molecule will be directly measured by hybridization with radioactively labeled cDNA probes obtained (a) via synthesis of 3 oligonucleotide probes and (b) via a new and more efficient cloning technique. After preliminary extraction of total RNA, hybridization to cDNA will be performed on nitrocellulose membranes. Where possible, in situ hybridization histochemistry will also be performed. Secretion will be studied: (1) by standardization of in vitro systems for studying release in rat and ovine tissues and (b) to examine release in situ, the reverse hemolytic plaque assay will be employed in conjunction with immunocytochemical techniques.